Hi my name is Evan Wu. I am currently living in Forest Hills, New York and attending Hunter College High School in Manhattan. I am a rising junior, and have taken Biology in my freshman year. Due to my long term interests in in science and in particular, the study of life, the coming year I plan to take AP Biology, as well as become even more active in my school's scientific community. I really like turtles (a lot!) and am often tired. I am excited to see what I will find during my 3 weeks at this camp.
Research: The Use of Bacterial Nanowires in Bioremediation and Electrical Systems
Background Respiration is a key process that happens in all living organisms, and is commonly seen as one of the identifying processes of life. Cellular respiration refers to the chemical pathways used in living cells to attain energy from food. It is separated into two distinct forms of respiration: aerobic (in the presence of oxygen) and anaerobic (without oxygen) respiration. In aerobic respiration, oxygen and glucose are taken in by the cell and turned into ATP. This is typically realized in the mitochondria of eukaryotic cells, with the processes of glycolysis, Kreb’s cycle, then oxidative phosphorylation.
Anaerobic respiration is more relevant to the topic at hand, as many bacteria function either partly or completely without oxygen, filling in certain environmental niches that allowed them to proliferate to this day and age. Anaerobic respiration replaces the oxygen as the electron acceptor in aerobic respiration with a substance such as ethanol, or nitrates and sulfates. These pathways are much less efficient compared to aerobic respiration (2 ATP with ethanol fermentation compared to the ideal 38 ATP produced through aerobic respiration).
A reduction site is crucial for the continuation of anaerobic respiration. Cellular survival depends on the 2 ATP produced from glycolysis to function. In glycolysis H2 is attached to 2 NAD+ in order break up glucose (6 carbons) into 2 pyruvate (3 carbons). The products of this reaction are 2 NADH and 2 ATP. Thus it, is very important to recycle NAD+ through oxidizing NADH, else the cell is depleted of its respiratory abilities and dies. The bacteria Shewanella oneidensis and Geobacter sulfurreducens achieve this necessary redox reaction by creating bacterial nanowires, which serve as external electron transfer pathways to facilitate anaerobic respiration. The nanowires created are characterized as specialized pili in Geobacter, but extensions of the membrane in the form of filaments dotted with cytochromes in Shewanella. In both cases, the nanowires were able to conduct an electrical signal (move electrons) outside of the cell, connecting the ethanol fermentation process with a reduction site such as solid Fe and Mn oxides, or graphite nodules. However, since Geobacter and Shewanella also partake in aerobic respiration, bacterial nanowires have only been observed when the bacteria were deprived of oxygen.
Fig. 3 Fig. 2
Cellular Systems for Diverse National Needs (<em>Shewanella</em>)
The form of externalized electron transfer in Geobacter and Shewanella differ in their methods. The nanowires of Geobacter are characterized as type IV pili, and they themselves have the ability to conduct electricity through metallic-like conduction. As the name suggests, the pili act as metal wires due to delocalized electron charges that arise from overlapping pi-bond orbitals of aromatic amino acids. This in essence creates a free sea of electrons which allows charges to move through the material. On the other hand, Shewanella are believed to utilize electron hopping through closely spaced cytochromes in order to transport its charges. Cytochromes are proteins that facilitate redox reactions and may also serve as points of electrical interactions in organisms. There is evidence that cytochromes are greatly associated with the conductivity of Shewanella filaments (through deleting the genes for cytochromes MtrC and OmcA), but the actual composition and mechanisms of the filaments are unknown.
The last thing to note in this background is the DIET (direct interspecies electron transfer) a relationship between two or more bacterial species which will probably be of importance in further applications and research. This syntrophic relationship is shown primarily with members of the Geobacter species. Instead of transferring electrons via bacterial nanowires to surrounding reduction sites, they are transferred to another surrounding species. An example of this would be the interaction between Geobacter metallireducens and Methanosaeta harundinacea, an abundant methanogen. Accepting electrons from G. metallireducens allowed M. harundinacea to process carbon dioxide to methane instead of acetate to methane. This syntrophy was also noted in other species, and the exact functions of how electrons are transferred or why are still under research.
Applications The sources suggested many different applications of harnessing the powers of bacterial nanowires, but not much has been developed due to knowledge constraints about the bacteria and nanowires themselves. However, many innovative and inspiring ideas are being developed and may see usage in the future.
1. Creating more efficient microbial fuel cells (MFCs). Fig. 6: Diagram showing the workings of a microbial fuel cell. Contains many similarities to hydrogen fuel cells. http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/Articleimage/2007/CP/b703627m/b703627m-f1.gif MFC utilize anaerobic bacterial activity to catalyze redox reactions between a man-made cathode and anode, creating an electrical current. The anode is typically an organic compound that is oxidized to produce protons and electrons, which are then used to reduce oxygen into water. It is a “clean” energy source, using sustainable and readily available products (organic waste, oxygen, graphite, etc.) and in many cases removing otherwise unusable organic waste. However, these fuel cells generate low current, and less efficient than similar methods, such as biogas power plants. Geobacter biofilms, with genetically enhanced nanowire pili, may be useful in replacing the anode terminal and increasing overall current production. They may also be useful in the construction of membranes, which are necessary in most designs to prevent oxygen from reaching the anode chamber, and interfering with anaerobic respiration. MFCs are inexpensive, and may be an important source of energy when enhanced with bacterial nanowires.
By applying the anaerobic respiration methods found with bacterial nanowires, we may be able to enhance or create new methods of energy creation or transportation. DIET has been shown to benefit or encourage faster methane production in methanogens. Methanogens are currently being used in biogas systems in order to convert organic waste into the more useful form of methane and carbon dioxide gas. The resulting gas can then be directly combusted, or processed into natural gases. DIET may also be an applicable process to anaerobic digesters outside of the methanogen group, possibly increasing functions in other similar bacteria. Microbial electrosynthesis is also a promising technique, which utilize bacteria to reduce CO2 and create organic products. If combined with solar energy, the process becomes a form of photosynthesis to create useful organic products. Bacterial nanowires may help catalyze the reduction necessary, but this requires growing thicker and more powerful biofilms. In addition, the ability of DMRBs (dissimilatory metal-reducing bacteria, i.e. bacteria with nanowires) to reduce and/or consume certain pollutants: human waste, animal waste, oils, compost, uranium, and gold also have various uses. This includes pollution control, wastewater purification (especially of uranium, which precipitates on the surface of the bacteria, removing it from from the system), a clean energy source, and even mining precious metals.
3. Using bacterial nanowires as microscopic-scale conductors Although tiny, bacterial nanowires have been shown to effectively transport electrons through relatively long distances. The fact that this conductivity is facilitated by proteins is also promising for future genetic engineering, and artificial construction of organic wires. DMRBs have been shown to produce thin, conductive biofilms, which supports using DMRBs as organic, microscopic wires. This may be relevant in creating bacterial computers, and other bioelectronics. As bacteria they may be able to proliferate at an efficient rate to replace conventional wiring. On the other hand, we may be able to harvest nanowires without having to use bacteria, allowing a new form of electrical conduction that may be more useful in oxygen containing environs. We may discover other organisms with electrically conductive filaments that serve other functions, possibly leading to innovation and advancements.
I. Purpose The purpose of this design is to create a new food preservation method that is both effective and convenient for usage in developing countries. Because developing countries may have little-to-no access to electricity, and may be unable to purchase refrigerators, other methods of food preservation are crucial to increasing food product yields in developing countries. Estimates claim that around 20% of food worldwide is lost due to food spoilage prior to consumption. In addition, most current research is focused on increasing food production rather than food preservation due to the workings of agribusiness. The problem with this is that technological advances in tend to increase yields only marginally, and may be threatening to the environment. In response, I propose creating easily growable strains of predatory bacteria, which will in theory help to eliminate microorganisms that cause food to spoil in a safe, convenient, and cheaper way.
II. Competing technologies Standard practices such as refrigeration are very effective in most developed countries, but this is not typically widespread in developing countries. Other methods include chemical preservation, drying, and canning; but most of these methods require facilities that are not typically available, or they may interfere with nutrient content and create less fresh or nutritionally inferior products.
III. The design Food spoils mostly due to the activities of various microorganisms within or on the surface of the food, that renders the food unsuitable for consumption. Bacteria, yeasts, and fungi are the main antagonists here. Spoilage bacteria digest food into waste products that are toxic or displeasing to eat, and consume nutrients within the food. This causes rancidity, slime formation, and soft rot, all of which may cause the person’s digestive system to reject the food. In addition, some of these bacteria may be pathogenic, causing food poisoning or diseases themselves e.g. E. Coli and salmonella. Yeasts digest food through the process of fermentation, creating lactic acid (sour spoiling) or sulfides (greening), compromising flavor and nutrient content. Molds make colonies on all varieties of foods, tending to grow in absence of spoilage bacteria in low pH environments. Mold digestion causes rancidity and taste changes in foods, as well as being harmful when ingested. There are many potential candidates for predator and prey. Utilizing different combinations under the same principle may be useful for treating a wider range of microorganisms. Examples follow:
Mold: Rhizopus → Fungal species that is associated with soft rot in fruits andvegetables, as well as colonies in bread and meat. Some species infect humans Predator: myxobacteria → certain species of myxobacteria can digest chitin andcellulose, which helps break down and digest fungal cell walls. Care must betaken since some species can also produce harmful toxins. Unknown inpathogenic in humans, more research and specification is necessary forutilization.
As an example, I will develop a design to consume bacteria of the Erwinia genus, a common Gram negative family that is present in vegetables. Since developing countries tend to get food from agriculture, eliminating Erwinia species may have significant benefits. In addition, members of Erwinia are related to E. Coli and Salmonella, and research has already found these pathogens susceptible to the selected predator: Bdellovibriobacteriovorus. Erwinia carotovora is a prominent species within the genus, known especially for inducing soft rot in important vegetables such as the potato. It infects fresh vegetables and breaks down cell walls via release of pectinase and cellulase. When breaking down pectin walls, galacturonic acid as well as other oligo uronides. Fig. 9: Diagram showing the life cycle of Bdellovibrio and its different tactics. https://microbewiki.kenyon.edu/images/thumb/f/f7/Bdellovibriolifecycle.png/800px-Bdellovibriolifecycle.png
Bdellovibrio will be equipped with protein receptors to sense galacturonic acid polymers, which allows specialized identification and targeting. This is realized through two component regulatory system; once galacturonic acid is recognized and Bdellovibrio is attached to a bacterial cell, it will release a mix of hydrolytic enzymes (serine, cysteine, aspartate, others) which allows it to penetrate the prey cell’s membrane. Bdellovibrio then begins the second part of its life cycle, absorbing nutrients from the prey grow in a specialized structure called the bdelloplast. Once it becomes sufficiently long and developed, the single Bdellovibrio cell septates into multiple progeny, that then burst out of the prey cell, leaving it dead, exhausted, and/or lacking a genome. Fig. 10: Initial state of design, where Erwinia has infected a potato and is breaking down the pectin in its cell walls into different sized polymers (or monomers) of galacturonic acid.
Fig. 11: This diagram shows the way Bdellovibrio will work to identify and infect Erwinia. Receptors on the surface of the membrane detect galacturonic acid and as such, it begins to produce hydrolytic enzymes. These enzymes will allow entry into the prey cell, which Bdellovibrio has attached to. Then, the natural life cycle of Bdellovibrio renders the Erwinia dead or dysfunctional.
The design can be made in the form of a bacterial spray in a water or aqueous solution, since Bdellovibrio move through swimming with their flagella. It can also be used in a sort of “bath,” in which food is placed in the liquid and preserved.
IV. Expected results When working ideally, we can expect the genetically modified B. bacteriovorus to be an effective E. carotovora eliminator. In addition, other Erwinia species who undergo the same process of galacturonic acid production may also be susceptible to targeted predation. This will eliminate or slow down the process of soft rot immensely in various fruits and vegetables. Bdellovibrio will proliferate by themselves, meaning little to no further maintenance.
Truth Table:
Galacturonic Acid Presence
Bdellovibrio Attached to Prey
Hydrolytic Gene Expression
0
0
0
0
1
0
1
0
0
1
1
1
V. Advantages This technology has the potential to be advantageous over existing technologies due to being easier to make, control, and operate. It is also evenly or possibly even more efficient than existing technology, as well as being cost efficient. As such, it may see use in developing countries, and while transporting products long distances. 20% of all food is estimated to be lost to spoilage by microorganisms prior to consumption, which has can support around 2 billion more people. This has the potential to be applied to other food diseases and blights, being able to target a variety of spoilage microorganisms depending on detecting their waste products.
VI. Potential Problems Bdellovibrio has been shown to almost exclusively target bacterial cells, and is very good at it. It ignores mammalian cells, which mitigates concerns for health risks, but it is also unable to target yeast and fungi, the other microorganisms involved. If the genetically engineered Bdellovibrio escapes into the environment, there could be unknown and possibly harmful effects to the ecosystem. The whole process must be streamlined, and inserting the genes necessary to create this system may have unintended effects in the bacterium itself. This may be solved through testing the product. It is also unlikely, but since Bdellovibrio in this design only targets cells after an infection, and does not outright kill prey cells (only impairs them greatly/fatally), it may be less effective than intended. In addition, since Erwinia have pathways to metabolize galacturonic acid, they may digest enough so that Bdellovibrio does not target it, though this is unlikely. Trial and error and resulting improvements to the system’s dynamics may help solve some of these concerns.
VII. Testing Testing can only really be effective through creating a prototype product and seeing whether it helps in preventing soft rot spoilage. Precautions must be made during testing not to allow the bacteria outside of the laboratory, since it may have unintended effects. Additionally, new discoveries concerning spoilage and predatory microorganisms can be made while improving the efficiency/potency of this technology. It has the potential to be applied to other diseases and blights, so looking at the real-life expression of the mechanics involved may be useful in developing further technologies. In the end, we may be just bathing foods in specialized baths full of unharmful predatory bacteria, which may see usage in developed countries in supermarkets. Supermarkets tend to leave fruits and vegetables out in the open, which may lead to faster spoilage.
Evan Wu
BLI Biological Research Session II
2016
Presentation:
https://docs.google.com/presentation/d/1sESALCYBSCZKlW3C1dri8XCKLRdLfd1cmnrqY9GmDfw/edit?usp=sharing
Research: The Use of Bacterial Nanowires in Bioremediation and Electrical Systems
Background
Respiration is a key process that happens in all living organisms, and is commonly seen as one of the identifying processes of life. Cellular respiration refers to the chemical pathways used in living cells to attain energy from food. It is separated into two distinct forms of respiration: aerobic (in the presence of oxygen) and anaerobic (without oxygen) respiration. In aerobic respiration, oxygen and glucose are taken in by the cell and turned into ATP. This is typically realized in the mitochondria of eukaryotic cells, with the processes of glycolysis, Kreb’s cycle, then oxidative phosphorylation.
Fig. 1
Fig. 1: Diagram showing glycolysis and fermentation. Glycolysis happens regardless of oxygen presence, and is the main source of energy derived from anaerobic respiration.
https://qph.ec.quoracdn.net/main-qimg-4bba0f0135b522aeb79bba9eaf3322cd?convert_to_webp=true
Anaerobic respiration is more relevant to the topic at hand, as many bacteria function either partly or completely without oxygen, filling in certain environmental niches that allowed them to proliferate to this day and age. Anaerobic respiration replaces the oxygen as the electron acceptor in aerobic respiration with a substance such as ethanol, or nitrates and sulfates. These pathways are much less efficient compared to aerobic respiration (2 ATP with ethanol fermentation compared to the ideal 38 ATP produced through aerobic respiration).
A reduction site is crucial for the continuation of anaerobic respiration. Cellular survival depends on the 2 ATP produced from glycolysis to function. In glycolysis H2 is attached to 2 NAD+ in order break up glucose (6 carbons) into 2 pyruvate (3 carbons). The products of this reaction are 2 NADH and 2 ATP. Thus it, is very important to recycle NAD+ through oxidizing NADH, else the cell is depleted of its respiratory abilities and dies. The bacteria Shewanella oneidensis and Geobacter sulfurreducens achieve this necessary redox reaction by creating bacterial nanowires, which serve as external electron transfer pathways to facilitate anaerobic respiration. The nanowires created are characterized as specialized pili in Geobacter, but extensions of the membrane in the form of filaments dotted with cytochromes in Shewanella. In both cases, the nanowires were able to conduct an electrical signal (move electrons) outside of the cell, connecting the ethanol fermentation process with a reduction site such as solid Fe and Mn oxides, or graphite nodules. However, since Geobacter and Shewanella also partake in aerobic respiration, bacterial nanowires have only been observed when the bacteria were deprived of oxygen.
Fig. 3 Fig. 2
Fig 2: Closeups showing a nanowire filament. Diameter of approx. 100 nm and height of 5-10 nm. Ridges can be observed, which are actually separate filaments http://www.pnas.org/content/103/30/11358.full
Fig 3: View of Shewanella and its nanowires. https://public.ornl.gov/site/gallery/detail.cfm?id=2&topic=&citation=&general=&restsection=
Fig. 4: View of Geobacter and its nanowires. http://www.asknature.org/strategy/af3010e709f572d5732c6a8c44c6eae2
The form of externalized electron transfer in Geobacter and Shewanella differ in their methods. The nanowires of Geobacter are characterized as type IV pili, and they themselves have the ability to conduct electricity through metallic-like conduction. As the name suggests, the pili act as metal wires due to delocalized electron charges that arise from overlapping pi-bond orbitals of aromatic amino acids. This in essence creates a free sea of electrons which allows charges to move through the material. On the other hand, Shewanella are believed to utilize electron hopping through closely spaced cytochromes in order to transport its charges. Cytochromes are proteins that facilitate redox reactions and may also serve as points of electrical interactions in organisms. There is evidence that cytochromes are greatly associated with the conductivity of Shewanella filaments (through deleting the genes for cytochromes MtrC and OmcA), but the actual composition and mechanisms of the filaments are unknown.
Fig. 5: Electron Hopping on top vs. Metallic-Like Conduction on bottom. Electrons can hop from cytochrome to cytochrome due to their charge conducting structure, but MLC relies on charges flowing through a chain of pi orbitals. https://encrypted-tbn1.gstatic.com/images?q=tbn:ANd9GcRqFeEtwDt_oXYf6yM4VbciWAl8SGZECeoT4ipzDXl4eeaKhK0a
The last thing to note in this background is the DIET (direct interspecies electron transfer) a relationship between two or more bacterial species which will probably be of importance in further applications and research. This syntrophic relationship is shown primarily with members of the Geobacter species. Instead of transferring electrons via bacterial nanowires to surrounding reduction sites, they are transferred to another surrounding species. An example of this would be the interaction between Geobacter metallireducens and Methanosaeta harundinacea, an abundant methanogen. Accepting electrons from G. metallireducens allowed M. harundinacea to process carbon dioxide to methane instead of acetate to methane. This syntrophy was also noted in other species, and the exact functions of how electrons are transferred or why are still under research.
Applications
The sources suggested many different applications of harnessing the powers of bacterial nanowires, but not much has been developed due to knowledge constraints about the bacteria and nanowires themselves. However, many innovative and inspiring ideas are being developed and may see usage in the future.
1. Creating more efficient microbial fuel cells (MFCs).
Fig. 6: Diagram showing the workings of a microbial fuel cell. Contains many similarities to hydrogen fuel cells. http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/Articleimage/2007/CP/b703627m/b703627m-f1.gif
MFC utilize anaerobic bacterial activity to catalyze redox reactions between a man-made cathode and anode, creating an electrical current. The anode is typically an organic compound that is oxidized to produce protons and electrons, which are then used to reduce oxygen into water. It is a “clean” energy source, using sustainable and readily available products (organic waste, oxygen, graphite, etc.) and in many cases removing otherwise unusable organic waste. However, these fuel cells generate low current, and less efficient than similar methods, such as biogas power plants. Geobacter biofilms, with genetically enhanced nanowire pili, may be useful in replacing the anode terminal and increasing overall current production. They may also be useful in the construction of membranes, which are necessary in most designs to prevent oxygen from reaching the anode chamber, and interfering with anaerobic respiration. MFCs are inexpensive, and may be an important source of energy when enhanced with bacterial nanowires.
2. Creating systems to convert organic waste, wastewater, etc. into electricity; bioremediation.
Fig. 7: Diagram showing the basic workings, inputs and outputs, of a biogas system https://www.americanbiogascouncil.org/images/genericDigestionProcess.gif
By applying the anaerobic respiration methods found with bacterial nanowires, we may be able to enhance or create new methods of energy creation or transportation. DIET has been shown to benefit or encourage faster methane production in methanogens. Methanogens are currently being used in biogas systems in order to convert organic waste into the more useful form of methane and carbon dioxide gas. The resulting gas can then be directly combusted, or processed into natural gases. DIET may also be an applicable process to anaerobic digesters outside of the methanogen group, possibly increasing functions in other similar bacteria. Microbial electrosynthesis is also a promising technique, which utilize bacteria to reduce CO2 and create organic products. If combined with solar energy, the process becomes a form of photosynthesis to create useful organic products. Bacterial nanowires may help catalyze the reduction necessary, but this requires growing thicker and more powerful biofilms. In addition, the ability of DMRBs (dissimilatory metal-reducing bacteria, i.e. bacteria with nanowires) to reduce and/or consume certain pollutants: human waste, animal waste, oils, compost, uranium, and gold also have various uses. This includes pollution control, wastewater purification (especially of uranium, which precipitates on the surface of the bacteria, removing it from from the system), a clean energy source, and even mining precious metals.
3. Using bacterial nanowires as microscopic-scale conductors
Although tiny, bacterial nanowires have been shown to effectively transport electrons through relatively long distances. The fact that this conductivity is facilitated by proteins is also promising for future genetic engineering, and artificial construction of organic wires. DMRBs have been shown to produce thin, conductive biofilms, which supports using DMRBs as organic, microscopic wires. This may be relevant in creating bacterial computers, and other bioelectronics. As bacteria they may be able to proliferate at an efficient rate to replace conventional wiring. On the other hand, we may be able to harvest nanowires without having to use bacteria, allowing a new form of electrical conduction that may be more useful in oxygen containing environs. We may discover other organisms with electrically conductive filaments that serve other functions, possibly leading to innovation and advancements.
Sources:
https://bio.as.uky.edu/sites/default/files/Current%20Opinion-Nanowires.pdf
http://www.pnas.org/content/103/30/11358.full
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663193/
http://web.mit.edu/pweigele/www/SoBEI/Info_files/Logan%202006%20Environ%20Sci%20Technol.pdf
https://www.americanbiogascouncil.org/biogas_what.asp
http://aem.asm.org/content/early/2014/05/12/AEM.00895-14.full.pdf+html
http://aem.asm.org/content/79/7/2397.full
https://www.sciencedaily.com/releases/2016/03/160324104809.htm
http://www.asknature.org/strategy/af3010e709f572d5732c6a8c44c6eae2
Design: Food Preservation Through Application of Genetically-Modified, Predatory Bacteria
Fig. 8: Picture of spoiled, mold and yeast infested tomatoes. http://c.fastcompany.net/multisite_files/codesign/imagecache/1280/poster/2012/11/1671202-poster-1280-1-joe-buglewicz-rotten.jpg
I. Purpose
The purpose of this design is to create a new food preservation method that is both effective and convenient for usage in developing countries. Because developing countries may have little-to-no access to electricity, and may be unable to purchase refrigerators, other methods of food preservation are crucial to increasing food product yields in developing countries. Estimates claim that around 20% of food worldwide is lost due to food spoilage prior to consumption. In addition, most current research is focused on increasing food production rather than food preservation due to the workings of agribusiness. The problem with this is that technological advances in tend to increase yields only marginally, and may be threatening to the environment. In response, I propose creating easily growable strains of predatory bacteria, which will in theory help to eliminate microorganisms that cause food to spoil in a safe, convenient, and cheaper way.
II. Competing technologies
Standard practices such as refrigeration are very effective in most developed countries, but this is not typically widespread in developing countries. Other methods include chemical preservation, drying, and canning; but most of these methods require facilities that are not typically available, or they may interfere with nutrient content and create less fresh or nutritionally inferior products.
III. The design
Food spoils mostly due to the activities of various microorganisms within or on the surface of the food, that renders the food unsuitable for consumption. Bacteria, yeasts, and fungi are the main antagonists here. Spoilage bacteria digest food into waste products that are toxic or displeasing to eat, and consume nutrients within the food. This causes rancidity, slime formation, and soft rot, all of which may cause the person’s digestive system to reject the food. In addition, some of these bacteria may be pathogenic, causing food poisoning or diseases themselves e.g. E. Coli and salmonella. Yeasts digest food through the process of fermentation, creating lactic acid (sour spoiling) or sulfides (greening), compromising flavor and nutrient content. Molds make colonies on all varieties of foods, tending to grow in absence of spoilage bacteria in low pH environments. Mold digestion causes rancidity and taste changes in foods, as well as being harmful when ingested.
There are many potential candidates for predator and prey. Utilizing different combinations under the same principle may be useful for treating a wider range of microorganisms. Examples follow:
Bacteria: Erwinia → G-, causes soft rot in fruits and vegetables. Extensive genus.
Pseudomonas → G-, sours and putrefies meats, fish, dairy, eggs. Somemembers pathogenic.
Proposed Predator: Bdellovibrio bacteriovorus → efficient G- predatory bacteria
Mold: Rhizopus → Fungal species that is associated with soft rot in fruits andvegetables, as well as colonies in bread and meat. Some species infect humans
Predator: myxobacteria → certain species of myxobacteria can digest chitin andcellulose, which helps break down and digest fungal cell walls. Care must betaken since some species can also produce harmful toxins. Unknown inpathogenic in humans, more research and specification is necessary forutilization.
As an example, I will develop a design to consume bacteria of the Erwinia genus, a common Gram negative family that is present in vegetables. Since developing countries tend to get food from agriculture, eliminating Erwinia species may have significant benefits. In addition, members of Erwinia are related to E. Coli and Salmonella, and research has already found these pathogens susceptible to the selected predator: Bdellovibriobacteriovorus. Erwinia carotovora is a prominent species within the genus, known especially for inducing soft rot in important vegetables such as the potato. It infects fresh vegetables and breaks down cell walls via release of pectinase and cellulase. When breaking down pectin walls, galacturonic acid as well as other oligo uronides.
Fig. 9: Diagram showing the life cycle of Bdellovibrio and its different tactics. https://microbewiki.kenyon.edu/images/thumb/f/f7/Bdellovibriolifecycle.png/800px-Bdellovibriolifecycle.png
Bdellovibrio will be equipped with protein receptors to sense galacturonic acid polymers, which allows specialized identification and targeting. This is realized through two component regulatory system; once galacturonic acid is recognized and Bdellovibrio is attached to a bacterial cell, it will release a mix of hydrolytic enzymes (serine, cysteine, aspartate, others) which allows it to penetrate the prey cell’s membrane. Bdellovibrio then begins the second part of its life cycle, absorbing nutrients from the prey grow in a specialized structure called the bdelloplast. Once it becomes sufficiently long and developed, the single Bdellovibrio cell septates into multiple progeny, that then burst out of the prey cell, leaving it dead, exhausted, and/or lacking a genome.
Fig. 10: Initial state of design, where Erwinia has infected a potato and is breaking down the pectin in its cell walls into different sized polymers (or monomers) of galacturonic acid.
Fig. 11: This diagram shows the way Bdellovibrio will work to identify and infect Erwinia. Receptors on the surface of the membrane detect galacturonic acid and as such, it begins to produce hydrolytic enzymes. These enzymes will allow entry into the prey cell, which Bdellovibrio has attached to. Then, the natural life cycle of Bdellovibrio renders the Erwinia dead or dysfunctional.
The design can be made in the form of a bacterial spray in a water or aqueous solution, since Bdellovibrio move through swimming with their flagella. It can also be used in a sort of “bath,” in which food is placed in the liquid and preserved.
IV. Expected results
When working ideally, we can expect the genetically modified B. bacteriovorus to be an effective E. carotovora eliminator. In addition, other Erwinia species who undergo the same process of galacturonic acid production may also be susceptible to targeted predation. This will eliminate or slow down the process of soft rot immensely in various fruits and vegetables. Bdellovibrio will proliferate by themselves, meaning little to no further maintenance.
Truth Table:
V. Advantages
This technology has the potential to be advantageous over existing technologies due to being easier to make, control, and operate. It is also evenly or possibly even more efficient than existing technology, as well as being cost efficient. As such, it may see use in developing countries, and while transporting products long distances. 20% of all food is estimated to be lost to spoilage by microorganisms prior to consumption, which has can support around 2 billion more people. This has the potential to be applied to other food diseases and blights, being able to target a variety of spoilage microorganisms depending on detecting their waste products.
VI. Potential Problems
Bdellovibrio has been shown to almost exclusively target bacterial cells, and is very good at it. It ignores mammalian cells, which mitigates concerns for health risks, but it is also unable to target yeast and fungi, the other microorganisms involved. If the genetically engineered Bdellovibrio escapes into the environment, there could be unknown and possibly harmful effects to the ecosystem. The whole process must be streamlined, and inserting the genes necessary to create this system may have unintended effects in the bacterium itself. This may be solved through testing the product. It is also unlikely, but since Bdellovibrio in this design only targets cells after an infection, and does not outright kill prey cells (only impairs them greatly/fatally), it may be less effective than intended. In addition, since Erwinia have pathways to metabolize galacturonic acid, they may digest enough so that Bdellovibrio does not target it, though this is unlikely. Trial and error and resulting improvements to the system’s dynamics may help solve some of these concerns.
VII. Testing
Testing can only really be effective through creating a prototype product and seeing whether it helps in preventing soft rot spoilage. Precautions must be made during testing not to allow the bacteria outside of the laboratory, since it may have unintended effects. Additionally, new discoveries concerning spoilage and predatory microorganisms can be made while improving the efficiency/potency of this technology. It has the potential to be applied to other diseases and blights, so looking at the real-life expression of the mechanics involved may be useful in developing further technologies. In the end, we may be just bathing foods in specialized baths full of unharmful predatory bacteria, which may see usage in developed countries in supermarkets. Supermarkets tend to leave fruits and vegetables out in the open, which may lead to faster spoilage.
Sources:
http://faculty.weber.edu/coberg/class/3853/3853%20MOs%20and%20Food%20Spoilage%20notes.htm
http://home.pacific.net.hk/~ppleung/Chem/microorganisms%20involved%20in%20food%20spoilage.htm
http://theplate.nationalgeographic.com/2014/07/31/the-world-food-preservation-center-solving-the-post-harvest-problem/
http://www.fao.org/docrep/t0073e/T0073E06.htm
http://home.pacific.net.hk/~ppleung/Chem/spoilage.htm
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3917220/
https://food-hygiene-essentials.com/691/pseudomonas-food-spoilage/
https://microbewiki.kenyon.edu/index.php/Erwinia_carotovora
http://www.mold.ph/rhizopus.htm
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908774/
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.623.2716&rep=rep1&type=pdf
http://science.sciencemag.org/content/303/5658/689.full
https://microbewiki.kenyon.edu/index.php/Bacteroides_thetaiotaomicron
http://1.bp.blogspot.com/-1aHyO5guOwA/U1FmysFk9KI/AAAAAAAAACU/MAqLvmPkt3k/s1600/spoil+orange.jpg
http://hort.uwex.edu/files/2014/11/Bacterial-soft-rot.jpg
http://www.foodengineeringmag.com/ext/resources/TECH_FLASH/TF-8-14-Spoiled-lemons422.jpghttp://www.foodengineeringmag.com/ext/resources/TECH_FLASH/TF-8-14-Spoiled-lemons422.jpg
http://www.all-science-fair-projects.com/project1605_108_1.html